In vitro evaluation on HeLa cells of protective mechanisms of probiotic lactobacilli against Candida clinical isolates.

AIMS: To characterize in vitro the ability of human Lactobacillus strains to inhibit the adhesion, to displace and to compete with clinically isolated Candida strains. METHODS AND RESULTS: Three types of assays were performed to determine the inhibitory effect of Lactobacillus plantarum 319, Lactobacillus rhamnosus IMC 501, Lactobacillus paracasei IMC 502 and a specific probiotic combination (SYNBIO) on adhesion of Candida pathogens to HeLa cells: blockage by exclusion (lactobacilli and HeLa followed by pathogens), competition (lactobacilli, HeLa and pathogens together) and displacement (pathogens and HeLa followed by the addition of lactobacilli). Bacterial adhesion to HeLa was quantified by microscopy after May-Grünwald/Giemsa stain. The inhibition results highlight a significant (P < 0·05) competition of the considered probiotics against all the Candida strains. The results suggest that the probiotic strains used in this study could prevent colonization of the urogenital tract by relevant pathogens such as Candida strains through barrier and interference mechanisms (mainly displacement and competition), but the degree of inhibition of adhesion was bacterial strain-dependent. CONCLUSIONS: The results support the potential of these Lactobacillus probiotic strains as anti-infective agents in the vagina and encourage further studies about their capacity to prevent and manage urogenital tract infections in females. SIGNIFICANCE AND IMPACT OF THE STUDY: To optimize the defensive properties of the vaginal microbiota, improving the health of many women by probiotic intervention.

Protective effect of Calamintha officinalis Moench leaves against alcohol-induced gastric mucosa injury in rats. Macroscopic, histologic and phytochemical analysis.

Calamintha officinalis Moench (Lamiaceae) is an aromatic plant used since ancient times for its preservative and medicinal properties. The plant, known as 'Mentuccia' in Central Italy, is used in cooking as an aromatizant and to impart aroma and flavour to food. The methanol extract of the leaves was subjected to phytochemical and biological investigations. The extract contains polyphenols, catechic tannins and terpenes and shows radical scavenger activity. By means of HPLC analysis, eriocitrin, eriodyctiol, acacetin, linarin, benzoic acid and some phenolic acids, such as caffeic, chlorogenic, p-coumaric, were determined. The gastroprotective activity of the extract was investigated using ethanol-induced ulcer in rats, with sucralfate as a reference drug. Samples of gastric mucosa, stained by PAS and haematoxylin/eosin, were observed by light microscopy. The efficacy of the extract was comparable to that of the reference drug. Probably the gastroprotective effect depends on a synergistic action of all the compounds occurring in C. officinalis leaves, even if the antioxidant potential of the leaves plays an important role by removing damaging agents from the gastric mucosa.

Glucan-associated protein modulations and ultrastructural changes of the cell wall in Candida albicans treated with micafungin, a water-soluble, lipopeptide antimycotic.

The composition of glucan-associated proteins (GAP) in the cell wall of Candida albicans was strongly affected by treatment with a sub-MIC yet beta-glucan synthesis inhibitory concentration (0.01 microg/ml) of FK463 (micafungin). Namely, a decrease in enzymes of glucose metabolism (mostly enolase and a novel 40 kDaltons component, here identified as the enzyme fructose-1,6-biphosphate aldolase) was observed, and this was coupled with an increase in two beta1-3 exo-glucanase isoforms (34 and 44 kDa, respectively). No GAP changes were detected in the same strain of the fungus made resistant to the drug, attesting to the specificity of the observed cell wall protein modulation. In addition, GAP changes were accompanied by marked ultrastructural alterations upon treatment with the sub-MIC dose of the drug, the majority of which was an aberrant cell surface morphology and a derangement of the normal layering of the cell wall. Our data demonstrate that sub-MIC doses of micafungin do critically affect not only the beta-glucan synthetic machinery but also protein composition and the whole cell wall structure of Candida albicans.

Generation of a recombinant 65-kilodalton mannoprotein, a major antigen target of cell-mediated immune response to Candida albicans.

A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogen Candida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designated CaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His(6)-tagged protein (rCaMp65) was expressed in Escherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4(+) T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.

In vitro activity of fluconazole and voriconazole against isolates of Candida albicans from patients with haematological malignancies.

We evaluated the activity of fluconazole and voriconazole against 83 Candida albicans isolates from patients with haematological malignancies, comparing the NCCLS microdilution method (M27-A) with a modified method with RPMI-2% glucose and MIC endpoint at 50% inhibition. Both drugs were highly active regardless of the year, the site of isolation of the strain and the test method employed. In several strains isolated during the last few years, trailing growth leading to difficulty in interpretation of the endpoint of the test has been observed for both drugs by the NCCLS method, but not by the modified method. In our experience, azole resistance of C. albicans is still not a clinical problem, however, the emerging phenomenon of the 'trailing effect' by the NCCLS method, even though resolvable by technical modifications, seems at least to indicate a reduction in the inhibitory activity of the azoles.

Biotyping and virulence properties of skin isolates of Candida parapsilosis.

The biotype and virulence of skin isolates of Candida parapsilosis were compared with blood isolates of the same fungus. Morphotype, resistotype, and electrophoretic karyotype determinations did not reveal any special cluster with a unique or dominant pathogenic feature among all of the isolates, regardless of their source. However, all cutaneous isolates had uniformly elevated secretory aspartyl-protease (Sap) activity, more than four times higher than the enzyme activity of the blood isolates. They were also highly vaginopathic in a rat vaginitis model, being significantly more virulent than blood isolates in this infection model. In contrast, skin isolates were nonpathogenic in systemic infection of cyclophosphamide-immunodepressed mice, while some blood isolates were, in this model, highly pathogenic (median survival time, 2 days, with internal organ invasion at autopsy). Finally, skin isolates did not differ, as a whole, from blood isolates in their adherence to plastic. This property was associated with a morphotype, as defined by a colony with continuous fringe, which was present among both skin and blood isolates. While confirming the genetic heterogenicity of C. parapsilosis, our data strongly suggest that the potential of this fungus to cause mucosal disease is associated with Sap production and is substantially distinct from that of systemic invasion.

High aspartyl proteinase production and vaginitis in human immunodeficiency virus-infected women.

Vaginal isolates of Candida albicans from human immunodeficiency virus-positive (HIV+) and HIV- women with or without candidal vaginitis were examined for secretory aspartyl proteinase (Sap) production in vitro and in vivo and for the possible correlation of Sap production with pathology and antimycotic susceptibility in vitro. HIV+ women with candidal vaginitis were infected by strains of C. albicans showing significantly higher levels of Sap, a virulence enzyme, than strains isolated from HIV+, C. albicans carrier subjects and HIV- subjects with vaginitis. The greater production of Sap in vitro was paralleled by greater amounts of Sap in the vaginal fluids of infected subjects. In an estrogen-dependent, rat vaginitis model, a strain of C. albicans producing a high level of Sap that was isolated from an HIV+ woman with vaginitis was more pathogenic than a strain of C. albicans that was isolated primarily from an HIV-, Candida carrier. In the same model, pepstatin A, a strong Sap inhibitor, exerted a strong curative effect on experimental vaginitis. No correlation was found between Sap production and antimycotic susceptibility, as most of the isolates were fully susceptible to fluconazole, itraconazole, and other antimycotics, regardless of their source (subjects infected with strains producing high or low levels of Sap, subjects with vaginitis or carrier subjects, or subjects with or without HIV). Thus, high Sap production is associated with virulence of C. albicans but not with fungal resistance to fluconazole in HIV-infected subjects, and Sap is a potentially new therapeutic target in candidal vaginitis.

Identification and modification of biphenyl dioxygenase sequences that determine the specificity of polychlorinated biphenyl degradation.

The polychlorinated biphenyl (PCB) congener specificities and partial BphA sequences of biphenyl dioxygenase were determined for a set of PCB-degrading bacteria. The strains examined were categorized into two groups based on their ability to degrade 17 PCB congeners. Strains that degraded a broad range of PCBs but had relatively weak activity against di-para-substituted PCBs were designated as having an LB400-type specificity. Strains designated as having a KF707-type specificity degraded a much narrower range of PCBs but had strong activity against certain di-para-substituted congeners. BphA protein sequence comparisons between these two types of strains identified four regions (designated I, II, III, and IV) in which specific sequences were consistently associated with either broad or narrow PCB substrate specificity. The dramatic differences in substrate specificity between LB400 and KF707 appear to result primarily from a combination of mutations in regions III and IV. Altering these regions in the LB400 BphA subunit to correspond to those in the KF707 sequence produced a narrow substrate specificity very similar to that of KF707. Some individual mutations within region III alone were found to improve PCB degradative activity, especially for di-para-substituted congeners. However, the greatest improvements in activity resulted from multiple amino acid modifications in region III, suggesting that the effects of these mutations are cooperative. These results demonstrate the ability to significantly improve PCB oxidative activity through sequence modifications of biphenyl dioxygenase.

Clinical and mycological evaluation of fluconazole in the secondary prophylaxis of esophageal candidiasis in AIDS patients. An open, multicenter study.

A prospective, multicenter, open study of fluconazole prophylaxis was performed in AIDS patients to evaluate the efficacy and toxicity of the drug in preventing relapses of esophageal candidiasis. To this aim, 99 AIDS patients who presented a first episode of clinically and microbiologically confirmed esophageal candidiasis were enrolled in eleven clinical centers scattered throughout the Italian territory. After resolution of this initial esophagitis, all subjects were given fluconazole, 100 mg/die, and followed up for a 6 month period. Only 7 out of the 99 patients enrolled had a relapse of Candida esophagitis, during a mean follow-up period of 138.5 days. All relapsing patients had CD4+ cell number < 100/microliters at baseline. Mild side effects were reported in only eight patients. However, 14 of the 27 subjects from whom serial serum samples were available became (12) or remained (2) antigenemic during fluconazole prophylaxis, independently from relapse, suggesting the persistence of tissue-invasive, proliferating Candida cells. Overall, the data of this study suggest a beneficial effect of prophylactic maintenance therapy with fluconazole against Candida esophagitis, particularly in the population with > 100 CD4+/microliters. However, the data on Candida antigenemia in these patients invite the consideration of a relative inefficiency of the drug to eradicate the microrganism from the esophageal tissue.

Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene.

Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners.

In situ stimulation of aerobic PCB biodegradation in Hudson River sediments.

A 73-day field study of in situ aerobic biodegradation of polychlorinated biphenyls (PCBs) in the Hudson River shows that indigenous aerobic microorganisms can degrade the lightly chlorinated PCBs present in these sediments. Addition of inorganic nutrients, biphenyl, and oxygen enhanced PCB biodegradation, as indicated both by a 37 to 55 percent loss of PCBs and by the production of chlorobenzoates, intermediates in the PCB biodegradation pathway. Repeated inoculation with a purified PCB-degrading bacterium failed to improve biodegradative activity. Biodegradation was also observed under mixed but unamended conditions, which suggests that this process may occur commonly in river sediments, with implications for PCB fate models and risk assessments.

Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400.

The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxygenase from Pseudomonas putida F1 and have been named bphA, bphE, bphF, and bphG. From this comparison, biphenyl dioxygenase was found to be a multicomponent enzyme containing a two-subunit iron-sulfur protein, a ferredoxin, and a reductase. Comparison of the large subunit of the iron-sulfur protein and the ferredoxin with other multicomponent dioxygenases identified amino acid sequences similar to Rieske iron-sulfur proteins for binding a [2Fe-2S] cluster. Sequences have also been identified in the reductase component that match the consensus sequence for FAD or NAD binding. Transcription of the biphenyl dioxygenase region was examined, and three transcription initiation sites were identified. Transcription initiating at the site furthest upstream is greatly increased when the LB400 cells are grown on biphenyl as the sole carbon source.

Vaginopathic and proteolytic Candida species in outpatients attending a gynaecology clinic.

Non-pregnant, non-diabetic outpatients were examined for the presence of pathogenic vaginal yeasts to determine if a correlation existed between a specific yeast and clinical disease. Yeasts were isolated as single vaginal species from 186 of 228 subjects with clinically diagnosed candidal vaginitis, as well as from 122 out of 380 asymptomatic, age-matched controls. Apart from Candida albicans and C glabrata, other prevalent species were C krusei, C parapsilosis and Saccharomyces cerevisiae which accounted for 9.2%, 6.0% and 5.4%, and 9.0%, 2.4% and 19.7%, of yeasts from patients and carriers, respectively. Only C albicans and C parapsilosis were significantly more common in those with vaginitis. Only the isolates of these two species secreted aspartyl proteinase in vitro, and the amount of the enzymes secreted by the isolates from patients was significantly higher than that secreted by the isolates from carriers. These two species consistently produced vaginal infection in pseudoestrus rats, whereas none of the non-proteolytic species tested (C glabrata, C krusei, and S cerevisiae) colonised the vagina in these rats. Proteinase secretion correlated with experimental vaginal infection; it could also be a reliable factor for distinguishing clinically active infection from asymptomatic fungal carriage.

The effect of antimycotics on secretory acid proteinase of Candida albicans.

The effect of antimycotics on secretory aspartate (acid) proteinase, a virulence enzyme of Candida albicans, was investigated. The conditions of the study were such as to induce proteinase production in the stationary phase of growth (25-40 hours), when no antifungal tested, except the polyene derivative methyl partricin, significantly reduced the viability of the culture. Among azole derivatives, fenticonazole (FZ) but not miconazole, fluconazole or ketoconazole, exerted strong inhibition on proteinase, in typical dose-diphasic pattern, (0.01 microgram/ml; 1-10 micrograms/ml). 5-fluorocytosine (5-FC) was also inhibitory at a dose interval 1-10 micrograms/ml. In all cases, the inhibition concerned the synthesis of the enzyme rather that its activity as suggested by the results of comparative ELISA, SDS-PAGE and spectrophotometric methods of proteinase detection. Finally, the inhibition of proteinase production by FZ and 5-FC mainly reflected the effect of these antimycotics on general protein synthesis.

Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation.

Pseudomonas strain LB400 is able to degrade an unusually wide variety of polychlorinated biphenyls (PCBs). A genomic library of LB400 was constructed by using the broad-host-range cosmid pMMB34 and introduced into Escherichia coli. Approximately 1,600 recombinant clones were tested, and 5 that expressed 2,3-dihydroxybiphenyl dioxygenase activity were found. This enzyme is encoded by the bphC gene of the 2,3-dioxygenase pathway for PCB-biphenyl metabolism. Two recombinant plasmids encoding the ability to transform PCBs to chlorobenzoic acids were identified, and one of these, pGEM410, was chosen for further study. The PCB-degrading genes (bphA, -B, -C, and -D) were localized by subcloning experiments to a 12.4-kilobase region of pGEM410. The ability of recombinant strains to degrade PCBs was compared with that of the wild type. In resting-cell assays, PCB degradation by E. coli strain FM4560 (containing a pGEM410 derivative) approached that of LB400 and was significantly greater than degradation by the original recombinant strain. High levels of PCB metabolism by FM4560 did not depend on the growth of the organism on biphenyl, as it did for PCB metabolism by LB400. When cells were grown with succinate as the carbon source, PCB degradation by FM4560 was markedly superior to that by LB400.

Sequence similarities in the genes encoding polychlorinated biphenyl degradation by Pseudomonas strain LB400 and Alcaligenes eutrophus H850.

DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to LB400 in PCB-degrading capability. These two organisms showed a strong conservation of restriction sites in the region of DNA encoding PCB metabolism. No other sequence similarities were detected in the two genomes. DNA from the other PCB-degrading strains showed no hybridization to the probe, which demonstrated the existence of at least two distinct classes of genes encoding PCB degradation.

Evidence for a correlation between proteinase secretion and vulvovaginal candidosis.

Candida albicans isolates from nondiabetic, nonpregnant outpatients with vaginitis were compared for in vitro proteinase secretion with isolates from women without specific candidal vaginitis symptomatology (carriers). Proteinase production was assayed in medium containing bovine hemoglobin (BH-P; 39 isolates in 69 independent determinations) or bovine serum albumin (BSA-P; 39 isolates in a single determination each). All isolates had measurable, consistent BH-P secretion, and most also showed detectable BSA-P activity. However, isolates from patients were more proteolytic than those from carriers, with the difference being statistically highly significant. When the patients with vaginitis were categorized according to signs and symptoms, the highest BH-P values were recorded for those with full symptomatology, whereas the only BSA-P-negative isolates were from the group without vaginitis. Isolates from the patient and carrier groups did not differ as a whole in their growth potential in vitro, and all were germ tube responders in serum, independent of their source.

Shigella sonnei endotoxin reduces arterial blood pressure but does not alter pituitary pro-opiocortin cleavage and corticosterone release in the rat.

Intravenous (iv) bolus injection of 320 and 640 micrograms/Kg of lipopolysaccharides (LPS) of S. sonnei (either in phase I or in phase II) resulted in a dose-dependent decrease in mean arterial pressure, when given in freely moving rats. No changes were observed in plasma beta-endorphin (beta-EP), ACTH and corticosterone levels. Pituitary beta-EP content was not modified 1 h after iv injection of LPS. Data suggest that doses of LPS, which are able to produce reversible hypotension, do not alter pro-opiocortin cleavage and corticosterone release.